JPH0717857A - Cardiovascular drug - Google Patents

Abstract

(57) [Abstract] [Objective] The present invention has an inhibitory action on Na ⁺ , K ⁺ -ATPase, an inhibitory action on binding to L-type calcium channel, an inhibitory action on c-AMP PDE and a blood viscosity lowering action. The object is to provide a compound useful as a therapeutic agent for diseases. [Structure] A medicinal licorice, a licorice which is its original plant, and a therapeutic agent for cardiovascular disease containing a compound isolated from other plants belonging to the same genus as an active ingredient.

Description

Detailed Description of the Invention

[0001]

INDUSTRIAL APPLICABILITY The present invention relates to Na ⁺ , K ⁺ -ATPase inhibitory action, L-type calcium channel binding inhibitory action, and c-AMP.
The present invention relates to compounds having a PDE inhibitory effect and a blood viscosity lowering effect, which are useful as therapeutic agents for cardiovascular diseases.

[0002]

2. Description of the Related Art In recent years, the number of patients with heart diseases such as heart failure and atrial arrhythmia has increased with the aging of the population, which has become a major social problem. In addition, the safe range of cardiac glycosides of the digitalis type widely used for these diseases is extremely narrow, and it is said that they are clinically difficult to use drugs, and development other than these drugs is desired. It was

Na ⁺ , K ⁺ -ATPase is an enzyme involved in active transport of Na ⁺ and K ⁺ , and inhibition of this enzyme causes Na ⁺ in cardiomyocytes to remain elevated. It is said that the Na ⁺ replaces Ca ⁺⁺ , the intracellular Ca ⁺⁺ concentration increases, contracts actomyosin, and enhances cardiomyocyte contraction.

[0004] Therefore, searching and development of cardiotonic drugs using Na ⁺ , K ⁺ -ATPase inhibitory action as an index have been carried out.

[0005] Calcium also plays an important role as a regulator of muscle contraction and cell movement in the living body. Calcium antagonists act on myocardium and smooth muscle, suppress the inflow of calcium from the outside of cells, cause relaxation of vascular smooth muscle, etc., and improve blood flow and decrease blood pressure, so they are used as clinical therapeutic agents. It is applied to vascular diseases such as hypertension, angina, arrhythmia, and cardiovascular disorders such as cerebral circulation disorder, and is expected to become more important in the future.

[0006] When considering the development of a new type of calcium antagonist, the binding
Since the assay (Nitrendipine Binding Assay, NTB) uses the inhibition of L-type calcium channel binding as an index, it serves as an important indicator of usefulness for the above-mentioned vascular diseases, and a compound having an action of inhibiting L-type calcium channel binding. Was required.

[0007]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive research to develop a drug for treating cardiovascular disease, and found that the herb licorice (Glycyrrhizae Radi
x), the licorice which is its original plant (Nishikita licorice, Tohoku licorice, Xinjiang licorice, etc.), compounds represented by the following formulas I to IV isolated from other homologous plants (dog licorice, etc.) are Na ⁺ , K ⁺ -ATPase inhibitory action, L-type calcium channel binding inhibitory action, c-AMP
They found that they have a PDE inhibitory action and a blood viscosity lowering action, and completed the present invention.

That is, the present invention is based on these findings and is as shown below.

Formula I below (However, R ₁ and R ₂ are the same or different and represent a hydrogen atom or a 3,3-dimethylallyl group.), The following formula II, Formula III below Or the following formula IV A therapeutic agent for cardiovascular disease, which comprises a compound represented by

Hereinafter, the compounds represented by the formulas I to IV are collectively referred to as the compound of the formula.

For example, the following method can be used to obtain the compound of the formula.

[0012] Crude licorice (Glycyrrhizae Radix), its original plant licorice (Seihoku licorice, Tohoku licorice, Xinjiang licorice, etc.), and other roots or whole plants of the same genus plant (dog licorice, etc.) may be added as necessary. After degreasing with hexane, extraction with an organic solvent such as benzene, ethyl acetate, ethanol, methanol, n-hexane, methanol, etc., the residue obtained by removing the solvent from the extract, methanol, benzene, ethyl acetate etc. Dissolved in a solvent, water, methanol, ethanol, acetic acid, chloroform, ethyl acetate, n-hexane, acetone, Amberlite XAD-2, Diaion HP-20, MCI gel as an elution solvent at least one selected from benzene, CHP20P
For example, porous polymer such as Sephadex LH-20, Sephadex such as Sephadex LH-20, reversed phase silica gel, silica gel, polyamide, activated carbon or cellulose column chromatography using preparative carrier, preparative thin layer chromatography, high performance liquid chromatography at least. It can be obtained by applying once.

In some cases, it may be purified by recrystallization using a suitable solvent such as methanol, ethyl acetate, benzene, ethyl ether, n-hexane, acetone, ethanol and the like.

The following are specific examples of the production of the compound of the formula.

Specific Example 1 6 kg of the above-ground portion of Tohoku licorice was extracted with ethanol, and the solvent was distilled off under reduced pressure to obtain 590 g of ethanol extract.

300 g of this ethanol extract was subjected to Amberlite XAD-2 column chromatography and eluted with a water-methanol-benzene system.

The benzene eluate was concentrated to obtain 25 g of a benzene fraction. 25 g of this benzene fraction was subjected to silica gel column chromatography to obtain n-hexane-
Elute with benzene and fraction from the benzene elution part
Got 1, 2 and 3.

Fraction 1 was subjected to Sephadex LH-20 column chromatography and recrystallized from benzene to obtain 18 mg of colorless needle crystals. The physicochemical properties of this colorless needle crystal are described in the literature [Phytochemistry, 30 , 1245 (1991)].
It was in agreement with that of Gankaonin S described.

Specific Example 2 Fraction 2 obtained in Specific Example 1 was subjected to high performance liquid chromatography and recrystallized from acetone-n-hexane to obtain 85 mg of colorless needle crystals. The physicochemical properties of this colorless needle crystal are described in the literature [Phytochemistry, 30 , 1245 (1991)].
It was in agreement with that of Gankaonin R described.

Example 3 Fraction 3 obtained in Example 1 was treated with Sephadex LH-2
By subjecting to 0 column chromatography and high performance liquid chromatography and recrystallizing from n-hexane-acetone, 70 mg of colorless needle crystals were obtained. The physicochemical properties of these colorless needles are described in the literature [Phytochemistry, 30 , 1245 (1991)].
Consistent with that of Gankaonin U described.

Specific Example 4 4.8 kg of northwest licorice was sequentially extracted with n-hexane, benzene and acetone. The solvent of the acetone extract was distilled off under reduced pressure to obtain 150 g of acetone extract. 150 g of this acetone extract,
Subjected to silica gel column chromatography, benzene
-Elution with an acetone system gave fractions 1 and 2 from the benzene-acetone (99: 1) eluate.

Fraction 2 was subjected to preparative thin layer chromatography and recrystallized from benzene-acetone to obtain 160 mg of colorless columnar crystals. The physicochemical properties of colorless needles was consistent with that of the literature [Heterocycles. 29 .1761 (1989) ] according Gurishiroru.

Specific Example 5 3 kg of the underground part of dog licorice was extracted with ethanol, and the solvent was distilled off under reduced pressure to obtain 190 g of ethanol extract.

190 g of this ethanol extract was subjected to Amberlite XAD-2 column chromatography and eluted with a water-methanol-benzene system.

The elution part of methanol was concentrated to obtain 60 g of a methanol fraction. 60 g of this methanol fraction
Was subjected to silica gel column chromatography and eluted with a benzene-methanol system to obtain Fraction 1 from the benzene elution part.

Fraction 1 was subjected to high performance liquid chromatography to obtain 50 mg of a colorless oily substance. Physicochemical properties of this colorless oil the literature [Heterocycles. 31 .643 (199
0)] The same as that of Gankaonin J described.

Next, the compound of the formula has an inhibitory action on Na ⁺ , K ⁺ -ATPase, an inhibitory action on binding to L-type calcium channel, and c-AMP.
The fact that it has a PDE inhibitory action and a blood viscosity lowering action and is useful as a therapeutic drug for cardiovascular disease will be described with reference to experimental examples.

Experimental Example 1 (Na ⁺ , K ⁺ -ATPase inhibitory action) In a test tube, 125 mM imidazole-hydrochloric acid buffer solution (pH 7.2) 200 μm
l, 1 M sodium chloride 50 μl, 200 mM potassium chloride 50 μl,
100 mg magnesium chloride 25 μl, 10 mM ethylenediaminetetraacetic acid dipotassium (EDTA ・ 2K) 25 μl, solution of compound of formula 50 μ
l, enzyme solution 50 μl (0.5 mg, Sigma, from Dog Kidne
y) was taken and incubated at 37 ° C for 20 minutes.

Next, 50 μl (1.35 mg, Sigma, from Equ) of the substrate, adenosine triphosphate disodium (ATP · 2Na), was added.
ine Muscle) was added and the reaction started. After incubating at 37 ° C for 20 minutes, add 2 ml of 20% trichloroacetic acid (TCA) to stop the reaction, leave it on ice for 10 minutes, and then use Fiske-Subbar.
Color was developed by the ow method, and inorganic phosphorus was quantified.

That is, 0.5 ml of 3% ammonium molybdate solution and 0.25% 1-amino-2-naphthol-4- were added to the reaction solution.
0.5 ml of a sulfonic acid (1,2,4-ANS) solution was added, the mixture was allowed to stand at room temperature for 15 minutes, centrifuged at 3,000 rpm for 5 minutes, and the absorbance at 660 nm was measured.

From this result, the inhibition rate (%) was calculated by the following equation.

[0032] A: Absorbance without compound of formula B: Absorbance with compound of formula

Inhibition rate (%) and inhibitory activity (IC
₅₀ ) is shown in Table 1.

Table 1

From the above results, the superior Na ⁺ ,
A K ⁺ -ATPase inhibitory effect was confirmed.

Experimental Example 2 (L-type calcium channel binding inhibitory action) (1) Preparation of rabbit skeletal muscle membrane protein After skeletal muscle was extracted from the thigh of the left hind limb of a 11-week-old Japanese white rabbit (Healthy), , Shredded with scissors. A 10-fold amount of 50 mM Tris-hydrochloric acid buffer (pH 7.4) was added thereto, and the mixture was homogenized with a Polytron homogenizer. Homogenate
Centrifuge at 5,000 xg for 20 minutes, then resuspend the supernatant at 45,000 x
Centrifuge for 30 minutes at g.

The precipitate was further centrifuged at 45,000 × g for 30 minutes. This precipitate was resuspended in 50 mM Tris-hydrochloric acid buffer solution so that the membrane protein concentration was 1 mg / ml, and used for the following experiments. All the above operations were performed at 4 ° C.

(2) [ ³ H] -Nitrendipine binding assay (NTB) 880 μl of 50 mM Tris-HCl buffer (pH 7.4), 100 μl of rabbit skeletal muscle membrane protein (1 mg / ml) in a test tube, compound of formula Solution of 10μ
l, [ ³ H] -Take 10 μl (3.7 kBq) of nitrendipine and at 25 ° C
Incubated for 1 hour.

After completion of the reaction, this reaction solution was subjected to Whatmann GF / B using a cell harvester (manufactured by BRANDEL).
Filter by suction with a filter, and filter with 5 ml ice-cold 50 mM.
The cells were washed twice with Tris buffer while aspirating. The filter was placed in a scintillation vial, and the radioactivity was measured with a liquid scintillation counter.

The inhibition rate (%) was calculated by the following formula.

[0041] A: Radioactivity without compound of formula B: Radioactivity with addition of compound of formula

Inhibition rate (%) and inhibitory activity (IC
₅₀ ) is shown in Table 2.

Table 2

From the above results, it was confirmed that the compound of the formula has an excellent inhibitory effect on the binding to the L-type calcium channel.

Experimental Example 3 (c-AMP PDE inhibitory action) 50 μl of 50 mM Tris-hydrochloric acid buffer solution (pH 7.5), 50 μl of 50 mM magnesium chloride, 50 μl of enzyme solution (Boehringer
Mannheim Yamanouchi Co., Ltd., beef heart), take the dimethylsulfoxide (DMSO) solution of the compound of formula, and add purified water to make the total amount.
The volume was adjusted to 450 μl and incubated at 37 ° C for 3 minutes.

Next, the substrate [ ³ H] -cyclic-AMP (0.01 mM,
The reaction was started by adding 50 μl of 1.0 × 10 ⁻⁶ dpm). 37 ° C
After incubating for 30 minutes in a test tube in a boiling water bath
The reaction was stopped by heating for 3 minutes. After cooling, snake venom (500 μg)
Was added and incubated at 37 ° C for 30 minutes.

After that, an ion exchange resin (Dowex-AG1-X8) was added, and the mixture was stirred and then centrifuged. The supernatant was taken and the radioactivity was measured with a liquid scintillation counter.

The inhibition rate (%) was calculated by the following formula.

[0049] C: Radioactivity without compound of formula S: Radioactivity with addition of compound of formula

The inhibition rates (%) of the compounds of the formula are shown in Table 3.

Table 3

From the above results, the excellent c-AM of the compound of the formula
A P PDE inhibitory effect was confirmed.

Experimental Example 4 (Blood viscosity lowering action) Male Wistar rats (10
Blood was collected from the abdominal aorta under ether anesthesia, and 40% ethylenediaminetetraacetic acid · 2 potassium [(EDTA · 2K) physiological saline] was added as an anticoagulant at a rate of 3 μl per 1 ml. Blood is centrifuged (3,000 rpm, 4 ° C, 5 minutes)
Then, the supernatant and the red blood cell layer were separated. The supernatant obtained by further centrifugation (3,000 rpm, 4 ° C, 15 minutes) was used as plasma. The hematocrit value (hereinafter referred to as the HT value) of the blood cell layer for several animals was measured, and the HT value was measured with plasma.
It was adjusted to 45% and used for blood viscosity measurement.

1 ml of blood for measurement was dissolved in 50% ethanol physiological saline at a final concentration of 4.8 × 10 ⁻⁴ M, and 50 μl of this solution was added to 1 ml of blood for blood viscosity measurement, and the mixture was incubated at 37 ° C. Incubated at C for 60 minutes. Next, 0.5 ml of the incubated blood was sampled and the viscosity was measured at a shear rate of 7.5 S ^-1 using a viscosity meter, and the degree of decrease in blood viscosity was calculated from the following formula.

[0055] A: Blood viscosity without compound of formula B: Blood viscosity with addition of compound of formula

The inhibition rates (%) of the compounds of the formula are shown in Table 4.

Table 4

From the above results, it was confirmed that the compound of the formula has an excellent effect of lowering blood viscosity.

Next, when an acute toxicity test of the compound of the formula was conducted using male ICR mice, it was a highly safe drug with no deaths after oral administration of 1.0 g / kg.

As described above, the compound of the formula has extremely low toxicity and high safety.

Next, the dose and formulation of the compound of formula will be explained.

The compounds of formula can be administered to animals and humans either neat or together with conventional pharmaceutical carriers. The dosage form is not particularly limited and may be appropriately selected and used as needed, and examples thereof include oral preparations such as tablets, capsules, granules, fine granules and powders, parenteral preparations such as injections and suppositories. To be

In order to exert a desired effect as an oral preparation, it depends on the patient's age, body weight and degree of disease, but usually 50 mg to 5 g as the weight of the compound of formula is divided into several times a day in adults. All doses seem appropriate.

Oral preparations include, for example, starch, lactose, sucrose,
Mannitol, carboxymethyl cellulose, corn starch, inorganic salts and the like are used in a conventional manner.

In this type of preparation, a binder, a disintegrating agent, a surfactant, a lubricant, a fluidity promoter, a flavoring agent, a coloring agent, a flavoring agent, etc. may be appropriately used in addition to the above-mentioned excipients. You can
Specific examples of each are as shown below.

[Binder] Starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, crystalline cellulose,
Ethyl cellulose, polyvinylpyrrolidone, macrogol.

[Disintegrant] Starch, hydroxypropyl starch, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, carboxymethyl cellulose, low-substituted hydroxypropyl cellulose.

[Surfactant] Sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polysorbate 8
0.

[Lubricant] Talc, waxes, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol.

[Flowability Accelerator] Light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate.

The compounds of formula can also be administered as suspensions, emulsions, syrups and elixirs, and these various dosage forms may contain flavoring agents and coloring agents. .

In order to exert a desired effect as a parenteral agent, it depends on the age, body weight and degree of disease of the patient.
In general, it is considered that intravenous injection, intravenous infusion, subcutaneous injection, and intramuscular injection of 0.1 mg to 1 g per day as the weight of the compound of formula in adults are considered appropriate.

This parenteral preparation is manufactured by a conventional method, and is generally used as a diluent in distilled water for injection, physiological saline, glucose solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol. Etc. can be used. Further, if necessary, a bactericide, a preservative, and a stabilizer may be added. Further, from the viewpoint of stability, this parenteral preparation may be filled in a vial or the like, frozen, and then water may be removed by an ordinary freeze-drying technique, and a liquid preparation may be re-prepared from the freeze-dried product immediately before use. Further, an isotonicity agent, a stabilizer, a preservative, a soothing agent and the like may be added as needed.

Other parenteral agents include external preparations, coating agents such as ointments, suppositories for rectal administration, and the like.
It is manufactured according to a conventional method.

Next, the formulation examples of the present invention will be described.

[Formulation Example 1]

According to the above formulation, the ingredients (1) to (4) were uniformly mixed and compression-molded with a tableting machine to give tablets (200 mg each).

Each tablet contains 20 mg of the compound obtained in Example 1, and 3 to 10 tablets for adults are to be taken in several divided doses per day.

[Formulation Example 2] Crystalline cellulose 84.5 g Magnesium stearate 0.5 g Carboxymethyl cellulose calcium 5 g Compound obtained in Example 2 10 g Total 100 g

According to the above formulation, a part of and was uniformly mixed, compression-molded, crushed, and the remaining amount of and was added and mixed, and compression-molded with a tableting machine to give one tablet.
0 mg tablets were obtained.

Each tablet contains 20 mg of the compound obtained in Example 2, and 3 to 10 tablets for adults are to be taken in several divided doses per day.

[Formulation Example 3] Crystalline cellulose 79.5 g 10% Hydroxypropyl cellulose ethanol solution 50 g Carboxymethyl cellulose calcium 5 g Magnesium stearate 0.5 g Compound 10 g obtained in Example 3 Total 145 g

According to the above formulation, and were uniformly mixed, the mixture was kneaded by a conventional method, granulated by an extrusion granulator, dried and crushed, and then mixed, and compressed by a tableting machine. It was molded to obtain a tablet of 200 mg each.

One tablet of the tablet contains 20 mg of the compound obtained in Example 3, and 3 to 10 tablets for adults are to be taken in several divided doses per day.

[Formulation Example 4]

According to the above prescription, the components (1) to (4) are uniformly mixed, compression-molded by a compression molding machine, and then crushed by a crusher,
Sieve to obtain granules.

1 g of this granule contains 100 mg of the compound obtained in Example 4, and 1 to 2 g of the daily dose for adults is divided into several doses.

[Formulation Example 5] Crystalline cellulose 86.5 g 10% Hydroxypropyl cellulose ethanol solution 35 g Compound obtained in Example 5 10 g Total 131.5 g

According to the above recipe, the ingredients (1) to (4) were uniformly mixed and kneaded. After granulating with an extrusion granulator, it was dried and sieved to obtain granules.

1 g of this granule contains 100 mg of the compound obtained in Example 5, and 1 to 2 g for an adult is to be taken in divided doses.

[Formulation Example 6] Corn starch 89.5 g Light anhydrous silicic acid 0.5 g Compound obtained in Example 1 10 g Total 100 g

According to the above formulation, were mixed uniformly, and No. 2 capsules were filled with 200 mg.

One capsule of this capsule contains 20 mg of the compound obtained in Example 1, and 3 to 10 capsules for an adult are to be taken in several divided doses per day.

[Formulation Example 7] Distilled water for injection 89.5 g Soybean oil 5 g Soybean phospholipid 2.5 g Glycerin 2 g Compound obtained in Example 2 1 g Total amount 100 g

According to the above-mentioned formulation, and were dissolved in, and the solution of and was added and emulsified to obtain an injection.

Claims (4)

[Claims] 1. The following formula I (However, R ₁ and R ₂ are the same or different and each represents a hydrogen atom or a 3,3-dimethylallyl group.) A therapeutic agent for cardiovascular disease comprising a compound represented by the formula (I) as an active ingredient. 2. The following formula II A therapeutic agent for cardiovascular disease, which comprises a compound represented by 3. The following formula III A therapeutic agent for cardiovascular disease, which comprises a compound represented by 4. The following formula IV A therapeutic agent for cardiovascular disease, which comprises a compound represented by